In situ antigen-generating cancer vaccine

ABSTRACT

The invention provides compositions and methods for utilizing scaffolds in cancer vaccines.

RELATED APPLICATIONS

This application is a national stage application, filed under 35 U.S.C.§371, of International Application No. PCT/US2012/040687, filed Jun. 4,2012, which claims the benefit of priority under 35 U.S.C. §119(e) toU.S. Provisional Application No. 61/493,398, filed Jun. 3, 2011, thecontents of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates generally to the field of cancer vaccines.

BACKGROUND OF THE INVENTION

Cancer accounts for approximately 13% of all human deaths worldwide eachyear. Existing dendritic cell-based therapeutic strategies are largelybased on ex vivo manipulation of dendritic cells to generate largenumbers of cells for activation with cancer antigen isolated from abiopsy of a patient's tumor. Because these ex vivo techniques areinvasive and expensive, there is a pressing need to develop dendriticcell-based cancer vaccine strategies that are not dependant on surgicalbiopsies and ex vivo manipulation of cells.

SUMMARY OF THE INVENTION

The invention represents a significant breakthrough in the treatment ofcancer in that tumor antigens for vaccination are generated withouthaving to take a biopsy from the patient, process the tumor cells exvivo, and then vaccinate the patient with processed tumor antigen. Onecan now achieve patient-specific cancer vaccines, without needingpatient-specific manufacturing of the vaccine, via the generation ofcancer antigens in situ using a scaffold that is implanted in the body.Live cancer cells are recruited to the 3-dimensional (3-D) vaccinescaffold following its placement in the patient, and the scaffold istreated to induce subsequent destruction of the recruited cancer cells.The destruction and/or lysis of the cancer cells generates antigens insitu in the scaffold.

Accordingly, the invention features a biopsy-free method for producing aprocessed (e.g., cell-dissociated) tumor antigen in situ. For example,the tumor antigen(s) are liberated from an intact tumor cell, associatedwith cell fragments, or associated with a cell that has been alteredfrom its naturally-occurring state. First, a porous 3-dimensionalscaffold is administered to a subject diagnosed with a cancer. Thescaffold comprises a chemoattractant of cancer cells. Such molecules(and their amino acid (aa) and nucleic acid (na) sequences) are wellknown in the art. For example, the chemoattractant of cancer cells is achemokine selected from the group consisting of chemokine (C-C motif)ligand 21 (CCL-21, GenBank Accession Number: (aa) CAG29322.1(GI:47496599), (na) EF064765.1 (GI:117606581), incorporated herein byreference), chemokine (C-C motif) ligand 19 (CCL-19. GenBank AccessionNumber: (aa) CAG33149.1 (GI:48145853), (na) NM_006274.2 (GI:22165424),incorporated herein by reference), stromal cell-derived factor-1 (SDF-1,GenBank Accession Number: (aa) ABC69270.1 (GI:85067619), (na) E09669.1(GI:22026296, incorporated herein by reference), vascular endothelialgrowth factor (e.g, VEGFA; GenBank Accession Number: (aa) AAA35789.1(GI:181971), (na) NM_001171630.1(GI:284172472), incorporated herein byreference), and interleukin-4 (IL-4, GenBank Accession Number: (aa)AAH70123.1 (GI:47123367), incorporated herein by reference).

The scaffold is maintained in situ for a time period sufficient toaccumulate circulating cancer cells, thereby yielding a cancercell-containing scaffold. Finally, the cell-containing scaffold iscontacted with a cytotoxic or cytolytic element to produce a processedtumor antigen. A cytotoxic or cytolytic element is a composition and/orcondition that causes death or lysis, respectively, of a cell. Forexample, the cell is a cancer cell. The patient to be treated comprisesa cancer that is characterized by circulating tumor cells, e.g.,metastatic tumor cells. For example, the subject is diagnosed with ametastatic cancer condition or a blood-borne cancer or cancer of thecirculatory system, e.g., leukemia. The cytotoxic or cytolytic elementcomprises a heat-conducting composition such as gold particles and/orthe application of external heat, ultrasound, laser radiation, or gammaradiation. For example, cytotoxicity or cytolysis of a cancer cell isinduced by applying a condition (e.g., an energy source such as thosedescribed above) to a cell-containing scaffold that also contains aheat-conducting composition. Suitable types of laser radiation includeultraviolet or near infrared laser radiation.

Exemplary scaffold compositions are described in US 2008-0044900 A1(incorporated herein by reference). Suitable scaffolds includepolylactic acid, polyglycolic acid, co-polymers of polylactic acid andpolyglycolic acid (e.g., PLGA polymers), alginates and alginatederivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin richgels, agarose, natural and synthetic polysaccharides, polyamino acids,polypeptides, polyesters, polyanhydrides, polyphosphazines, poly(vinylalcohols), poly(alkylene oxides), poly(allylamines)(PAM),poly(acrylates), modified styrene polymers, pluronic polyols,polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymersor graft copolymers of any of the above. One preferred scaffoldcomposition includes an RGD-modified alginate.

The scaffold composition is between 0.01 mm³ and 100 mm³. For example,the scaffold composition is between 1 mm³ and 75 mm³, between 5 mm³ and50 mm³, between 10 mm³ and 25 mm³. Preferably, the scaffold compositionis between 1 mm³ and 10 mm³ in size.

The porosity of the scaffold influences ingress/egress of the cells fromthe device. Pores are nanoporous, microporous, or macroporous. Theporous polymer device contains aligned and/or interconnected pores tofacilitate movement of cells into and out of the device. For example,immune cells such as DCs are recruited into the device, pick up antigen,e.g., antigen that has been liberated from cancer cells that have beenattracted to the device, and then migrate out of the device via theinterconnected pores to leave the device and go to other sites in thebody such as draining lymph nodes. For example, the diameter ofnanopores are less than about 10 nm; micropores are in the range ofabout 100 nm-20 μm in diameter; and, macropores are greater than about20 μm (preferably greater than about 100 μm, 200 μm, 300 μm and evengreater than about 400 μm). In one example, the scaffold is macroporouswith aligned or interconnected pores of about 400-500 μm in diameter.

Optionally, the scaffold further comprises a hyperthermia-inducingcomposition. Suitable hyperthermia-inducing compositions include amagnetic nanoparticle or a near infrared (NIR) absorbing nanoparticle.In some cases, the nanoparticle is magnetic, and the method furthercomprises contacting the magnetic nanoparticle with an alternativemagnetic field (AMF) to induce local hyperthermia in situ, therebyaltering or disrupting the cancer cell and producing a processed tumorantigen. In another example, the method further comprises contacting theNIR nanoparticle with NIR radiation to induce local hyperthermia insitu, thereby altering or disrupting the cancer cell and producing aprocessed tumor antigen. Hyperthermia is characterized by a localtemperature of greater than 37 degrees Celsius. For example, thetemperature of the device is temporarily heated to 40, 45, 50, 60, 70,75, 80, 85, 90, 95 or more degrees.

The size of the particles is tailored to the scaffolds of the invention.For example, the nanoparticle comprises a diameter of less than 200 nm,e.g., a diameter of greater than 2 nm and less than 150 nm, e.g., adiameter of 5-100 nm, e.g., a diameter of 10-50 nm. Exemplary particlesare less than 45 nm, e.g., 40 nm, or less than 15 nm, e.g., 13 nm. Asuitable NIR nanoparticle includes a gold nanorod, gold nanoshell,silica nanoparticle, gold nanocage, noble metal nanoparticle, carbonnanotube, carbon nanoparticle, and graphite nanoparticle.

The methods described herein are useful in the treatment of cancer in amammal. The mammal can be, e.g., any mammal, e.g., a human, a primate, amouse, a rat, a dog, a cat, a horse, as well as livestock or animalsgrown for food consumption, e.g., cattle, sheep, pigs, chickens, andgoats. In a preferred embodiment, the mammal is a human.

The invention also provides a tumor antigen-processing device comprisinga porous polymer, a chemoattractant for cancer cells, and acell-altering or cell-destroying (e.g., cytotoxic or cytolytic)composition or element such as a hyperthermia-inducing particle. Ahyperthermia-inducing nanoparticle is one that heats the cells withinthe scaffold to a cell-destructive temperature upon the application ofan external energy source. For example, the energy source is a form ofradiation such as heat, AMF or NIR.

An exemplary device comprises an immune cell (e.g., DC) recruitmentcomposition such as granulocyte macrophage colony-stimulating factor(GM-CSF). In one example, the chemoattractant, cytoxicity- orcytolysis-composition, and immune cell recruitment composition areinterspersed throughout the porous polymer. In another example, theporous polymer comprises a first zone comprising the chemoattractant andcytoxicity-inducing or cytolysis-inducing composition and a second zonecomprising the immune cell recruitment composition. In the latterexample, the zones are layered or constructed with a core-shellarchitecture, whereby the first zone is configured as a core and thesecond zone is configured as a shell. Exemplary cytotoxicity-inducing(or cytolysis-inducing) compositions are described above, e.g.,hyperthermia-inducing particles.

As used herein, an “isolated” or “purified” nucleotide or polypeptide(e.g., a chemoattractant, cytokine, or chemokine nucleotide orpolypeptide) is substantially free of other nucleotides andpolypeptides. Purified nucleotides and polypeptides are also free ofcellular material or other chemicals when chemically synthesized.Purified compounds are at least 60% by weight (dry weight) the compoundof interest. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight thecompound of interest. For example, a purified nucleotides andpolypeptides, e.g., a chemoattractant, cytokine, or chemokine is onethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w)of the desired oligosaccharide by weight. Purity is measured by anyappropriate standard method, for example, by column chromatography, thinlayer chromatography, or high-performance liquid chromatography (HPLC)analysis. The nucleotides and polypeptides are purified and used in anumber of products for consumption by humans as well as animals, such ascompanion animals (dogs, cats) as well as livestock (bovine, equine,ovine, caprine, or porcine animals, as well as poultry). “Purified” alsodefines a degree of sterility that is safe for administration to a humansubject, e.g., lacking infectious or toxic agents.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

Small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic and inorganic compounds (including heterorganic andorganomettallic compounds) having a molecular weight less than about5,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 2,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a sufficient amountof the formulation or component to provide the desired effect. Forexample, “an effective amount” of a chemoattractant of cancer cells isan amount of a compound required to mediate an accumulation of two ormore cancer cells in the scaffold device prior to application of acell-altering or cell-destroying stimulus. Ultimately, the attendingphysician or veterinarian decides the appropriate amount and dosageregimen.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. The terms “preventing” and“prevention” refer to the administration of an agent or composition to aclinically asymptomatic individual who is susceptible or predisposed toa particular adverse condition, disorder, or disease, and thus relatesto the prevention of the occurrence of symptoms and/or their underlyingcause.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps and permits those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, Genbank/NCBI accession numbers, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of recruitment of peripheral dendritic cells (DCs)to the scaffold, loading of recruited DCs with cancer antigen, and DCmaturation (induced by danger signals) such as CpG oligodeoxynucleotides(CpG-ODN)/ poly(ethyleneirnine) (PEI), e.g., PEI-condensed CpG ODN.

FIG. 2 is a diagram of circulating endogenous cancer cells beingrecruited to the vaccine scaffold following implantation in the patient,subsequent destruction of the recruited cancer cells leading toliberation of tumor antigens in situ in the scaffold, and DCactivation/loading with liberated tumor antigen.

FIG. 3 is a bar graph showing migration of leukemia cells in response toa gradient of CCL-21.

FIG. 4 is a bar graph showing in vivo recruitment of leukemic cells toblank and loaded scaffolds.

FIG. 5 is a photograph of explanted scaffolds retrieved from miceshowing fluorescent-nano particle (NP)-labeled leukemic cells that wererecruited into the scaffold by CCL-21.

FIG. 6A is a photograph of a scaffold in which gold nanorods (GNRs) wereincorporated in poly(lactide and glycolide) (PLG) macroporous scaffold(GNR-PLG scaffold).

FIG. 6B is a photomicrograph illustrating a microscopic view of themacroporous scaffold structure. The bar scale in the lower left-handcorner is 200 μm.

FIG. 6C is a photomicrograph showing gold nanorods (GNRs) coated withpoly(ethylene glycol) (PEG).

FIG. 7A is a line graph showing the intrinsic maximum absorption of GNRsincorporated in the PLG scaffold.

FIG. 7B is a photomicrograph of a thermal image of a GNR-PLG scaffoldafter irradiation with 808 nm continuous diode laser.

FIG. 8A is a line graph demonstrating the temperature of GNR-PLGscaffolds after application of a different laser power.

FIG. 8B is a line graph demonstrating that repetitive near infrared(NIR) irradiation allowed multiple hyperthermia in the same GNR-PLGscaffold.

FIG. 9 is a photomicrograph of a thermal image of a GNR-PLG scaffoldphysically attached to a half blank PLG scaffold and irradiated with 808nm laser.

FIG. 10A is a photograph of a single PLG scaffold composed of aGNR-incorporated core.

FIG. 10B is a photograph of a thermal image of a single PLG scaffoldcomposed of a GNR-incorporated core.

FIG. 10C is a photomicrograph showing the well-interconnected porestructure of a PLG scaffold.

FIG. 11A is a bar chart illustrating heat shock protein (HSP) levels ofcancer cell cultures after heat shock in 43° C.

FIG. 11B is a bar chart demonstrating in vitro DC activation byheat-shocked lysate and freeze-thaw lysate.

FIG. 12A is an illustration of cells (leukemic cells) in a GNR-PLGscaffold and irradiated with a laser (808 nm).

FIG. 12B is a bar chart showing the level of HSP70 in cancer cells afterexposure to various temperatures.

FIG. 12C is a photograph of a western blot showing that 24 hourincubation after irradiation at 45° C. generated the highest HSP levels.

FIG. 13 is a bar chart representing the relative cell population in aGNR-PLG scaffold with activated DC cell surface markers after NIRirradiation at 45° C.

FIG. 14A is a photograph of a thermal image of an NIR irradiated GNR-PLGscaffold seeded with leukemic cells.

FIG. 14B is a bar chart showing leukemic cell viability in a GNR-PLGscaffold after application of NIR irradiation.

FIG. 15A is a photograph of a thermal image of an NIR irradiated GNR-PLGscaffold seeded with leukemic cells subcutaneously implanted in aC57BL/6J mouse.

FIG. 15B is a bar graph of a bar chart showing in vivo leukemic cellviability in a GNR-PLG scaffold after application of NIR irradiation.

FIGS. 16A-B are bar graphs showing DC recruitment and activation.

FIGS. 17A-B are bar graphs showing draining lymph node cell number andDC activation in draining lymph node tissue.

FIGS. 18A-D are bar graphs showing DC recruitment and activation in PLGvaccine using laser irradiation.

FIG. 19 is a bar graph showing DC activation with heat-shocked cancercell lysate compared to freeze/thaw cancer cell lysate.

FIGS. 20A-B are scatter plots, and FIGS. 20C-D are bar graphs showing DCresponsiveness to lipopolysaccharide (LPS).

FIG. 21A is a photograph of an antigen-generating cancer vaccine devicewith a core-shell architecture.

FIG. 21B is a bar graph showing the Young's modulus characteristics ofan antigen-generating cancer vaccine with monophasic, layered, andcore-shell architecture.

FIG. 21C is a series of photographs of an antigen-generating cancervaccine scaffold device. The top panel shows the results of FLIR thermalimaging (thermography) of the device, and the bottom panel shows theresults of visible imaging (photography).

DETAILED DESCRIPTION

Three dimensional (3D) scaffolds provide a temporary residence fordendritic cells (DCs) and effectively regulate host DC trafficking andactivation in situ, while simultaneously preventing upregulation of thetolerizing arm of the immune system, and provide therapeutic protectionagainst cancer. For example, in the cancer vaccine systems describedherein, implantation of macroporous poly(lactide and glycolide) (PLG)scaffolds loaded with chemoattractant (GM-CSF) of DCs, cancer antigens(tumor lysates), and danger signals (CpG oligonucleotide) resulted inrecruitment of peripheral DCs to the scaffold, loading of recruited DCswith cancer antigen, and their maturation by danger signals (FIG. 1).The antigen-presenting mature DCs moved to lymph nodes and generatedpotent cytotoxic T lymphocyte (CTL) responses. In this manner, thisvaccine system triggered a strong anticancer immune response, whichallowed the eradication of the cancer. One such system utilizes a tumorbiopsy from the patient to be treated to generate the antigen, whichrequires ex vivo manipulation and processing of tumor tissue. Inaddition, this system requires that each vaccine be manufactured for thespecific individual to be treated (using the tumor lysate from that samepatient). The methods of the present invention represent an improvementof the heretofore-described system.

As described herein, a patient-specific anti-tumor immune response and areduction in tumor burden is achieved, without patient-specificmanufacturing of the vaccine. Instead, cancer antigens are generated insitu in a polymeric scaffold that was implanted in the body. Live cancercells present in the circulatory system of the subject are recruited tothe vaccine scaffold following its placement in the patient, and thesubsequent destruction of the recruited cancer cells generates antigensin situ in the scaffold (FIG. 2).

Recruitment of Circulating Cancer Cells to Scaffolds

Suitable cancers to be treated in this manner are those in which thereare circulating primary or metastatic cancer cells in the blood stream.These cells are characterized by migratory properties in response togradients of specific chemokines Thus, circulating cancer cells arerecruited to implanted scaffolds in which specific chemokines have beenincorporated.

-   -   Circulating cancer cells: metastatic cancer cells in various        cancers, leukemic cells.    -   Chemoattractant of cancer cells: various chemokines depending on        cancer type. (e.g., CCL-21, CCL-19, SDF-1, VEGF, IL-4 etc.).    -   3D scaffolds: various types of 3D scaffolds designed to have        pores and to load chemokines including biodegradable porous        polymer, porous inorganic materials, assembled nanoparticles,        nanotubes, nanorods, etc.        Destruction of the Recruited Cancer Cells by External Stimuli on        Scaffolds

As described below, in addition to recruitment of cancer cells toscaffolds, various external stimuli are used to kill the cancer cellsafter they are recruited in order to generate lysates containing cancerantigens in situ. External stimuli applicable to implanted scaffolds tokill the recruited cancer cells.

-   -   External Heating    -   Ultrasound    -   Laser irradiation: UV, Near infrared laser    -   Gamma irradiation    -   Nanoparticle (NP)-mediated hyperthermia        -   Alternative magnetic field (AMF) for scaffold loaded with            magnetic nanoparticles.        -   Near infrared (NIR) irradiation for scaffold loaded with NIR            absorbing nanoparticles (e.g., gold nanorods, gold            nanoshells, gold nanocages, other noble metal nanoparticles,            carbon nanotubes, carbon nanoparticles, graphite, etc.)            Separating the Manipulation of Recruited DCs and Cancer            Cells

Sometimes it is desirable to separate the recruited DCs and cancer cellsso the signals used to kill the cancer cells do not negatively impactthe DC functions. As described below, this segregation is accomplishedvia control over the temporal order of recruitment of each cell type, ora spatial segregation of the cells that allows application of externalstimuli to a specific region of scaffold.

-   -   Temporal control of the order of cell recruitment        -   Rather than recruiting all cells simultaneously, cancer            cells are first recruited and destroyed to generate a            lysate. Subsequently, cancer cells recruit DCs to the site            of the cancer antigens without damaging DCs by external            stimuli. For this purpose, it is possible to control of the            release profiles from scaffolds of different            chemoattractants to DCs and cancer cells. For example,            different composition of polymers with different degradation            profiles and/or different molecular affinities to each            chemokine are used in the preparation of a polymer vaccine            scaffold to control release profiles.    -   Spatial control of scaffolds to allow applying external stimuli        to specific region of scaffold        -   Scaffolds are also compartmentalized such that only certain            compartments are affected by the external stimuli to            specifically kill the cancer cells residing in those            compartments, thereby allowing for the maintenance of intact            and functional DCs in other compartments. For this purpose,            various structural modifications of scaffolds are utilized.            In the case of nanoparticle (NP)-mediated hyperthermia for            killing of cancer cells, NPs are incorporated in specific            regions of the scaffolds, which allows specific hyperthermia            in the NP-region and has a trivial hyperthermic effect to            other regions where DCs are recruited.            In situ Antigen-generating Cancer Vaccine

Immunotherapy with protein drugs (e.g., cytokines and monoclonalantibodies) is one approach for cancer management. Therapeutic cancervaccines, another form of immunotherapy, represent another approach totreat cancer. Cancer vaccines are designed to invoke strong anti-tumorimmune activity, and the induction of antigen-specific cytotoxic (CD8+)T lymphocytes (CTLs) is a critical aspect of their function. ActivatedCD8+ T cells kill tumor cells upon recognition of specific labels(antigens) present on tumor cells, and this recognition is dependent onbinding of the label to a T cell receptor (TCR) specific to thatantigen. Dendritic cells (DCs) are the most important antigen presentingcells (APCs), and play a key role in initiating CTL responses.

Prior to the invention described herein, the first DC-based therapeuticcancer vaccine, known as Provenge, was approved by the Food and DrugAdministration. This breakthrough in cancer therapy demonstrated thatthe stimulation of a patient's own immune system to fight cancer.However, this therapy is based on ex vivo manipulation of DCs in orderto generate large numbers of these cells, and to activate the cells withcancer antigen, and thus suffers from a high cost and significantregulatory burden. In addition, tumors were not eradicated with thistherapy, and the increase in patient survival time has been limited to 4months. While this breakthrough may have a major impact on cancertreatment, it also highlights the need to make further progress on theDC-based cancer vaccine strategy, and to bypass its dependency on exvivo manipulation.

Developments in material science have led to new biomaterials and theapplications of materials in a wide range of biomedical applications,including diagnostics, cancer therapy, and tissue regeneration. Inparticular, nanoparticles and macroscopic, three-dimensionalbiomaterials have significant potential in many clinical applications.As described herein, because of their nanosize and easy surfacemodification, targeting of nanoparticles to various tissues, includingtumors and lymph nodes is exploited to deliver imaging or therapeuticmodalities. 3-D macroscale biomaterials, especially porous scaffolds,have been extensively explored for applications involving the controlledrelease of growth factors, cell delivery, and tissue regeneration. Thesematerials create microenvironments that allow the fate of resident cellsto be modulated, typically via control over the physical properties andpresentation of cell signaling molecules from the walls of thematerials. These 3-D macroscale materials and nanoparticles are usefulin the development of vaccines in the context of cancer, particularlyvia the targeting and programming of specific immune cell populations.

As described in the examples below, porous polymer matrices that providea temporary residence for DCs effectively regulate host DC traffickingand activation in situ, while simultaneously preventing upregulation ofthe tolerizing arm of the immune system, and provide therapeuticprotection against cancer. Macroporous PLG scaffolds incorporating i)GM-CSF to recruit DCs, ii) CpG/PEI complex to mature the DCs, and iii)tumor lysate to provide a mixture of cancer antigens were developed forthis purpose. Upon subcutaneous implantation, GM-CSF was released andestablished a gradient in the surrounding tissue to recruit significantnumbers of host DCs. The presentation of CpG/PEI complex from thepolymer to the recruited DCs increased the maturation of DCs in thescaffolds and their LN-homing. These scaffolds induced strong specificCTL responses to melanoma in a prophylactic model, with a 90% survivalrate as well as in therapeutic models of melanoma and glioblastoma withover a 50% survival rate after vaccinations. This system recruitedvarious DC subsets, including significant numbers of plasmacytoid DCs(pDCs) and CD8+ DCs, which are very important in antigen crosspresentation, and the numbers of these DC subsets strongly correlatedwith the vaccine efficacy. This vaccine also diminished the localconcentrations of tolerogenic cytokines (e.g., IL-10, TGF-β), andnumbers of T regulatory cells, suggesting that a key aspect of itssuccess related to its ability to down-regulate tolerance. These effectswere only found when the polymer had the physical form of a macroporousscaffold, as the vaccine effectiveness was significantly diminished whenpolymer microspheres were used instead to provide a sustained, localizedrelease of the bioactive agents, without providing a residence for therecruited cells. This result indicates that creating a microenvironmentin which host environmental cues are minimized, and exogenous maturationfactors are highly concentrated, is a key to reprogram immune responsesin situations such as cancer where there exist significant,pathology-associated tolerizing cues.

However, a limitation in this system is that it requires a tumor biopsyfrom patients and ex vivo manipulation and processing to generate cancerantigens. A system to generate cancer antigens in situ in the scaffoldimplanted in the body without biopsy or any ex vivo manipulation ofcells represents an improvement over earlier systems. To make apatient-specific cancer vaccine, an improved scaffold system wasdeveloped in which cancer cells are recruited to a 3D vaccine scaffoldand the alteration or destruction of those recruited cancer cellsgenerates cell lysates in situ in the scaffold.

EXAMPLE 1 In situ Antigen-Generating Cancer Vaccine by Recruiting CancerCells and Subsequent Destruction of the Recruited Cancer Cells byExternal Stimuli

Described below are examples of gold nanorod-loaded cancer vaccinescaffolds to recruit leukemic cells and their subsequent destruction viaNIR irradiation-mediated hyperthermia to generate cancer antigen coupledwith heat shock protein. To demonstrate the scaffolds are capable ofrecruiting cancer cells, mouse leukemic cells (C1498) were tested intranswell assay using CCL-21 as the chemoattractant. C1498 showed strongmigration to the gradients of CCL-21 (FIG. 3).

EXAMPLE 2 In vivo Recruitment of Leukemic Cells

In vivo recruitment of leukemic cells was characterized usingGFP-expressing leukemic cells. PLG scaffolds without any chemokines(Blank), loaded with GM-CSF (G), and loaded with GM-CSF and CCL-21 (G+C)were implanted to C57BL/6J mice subcutaneously and GFP-leukemic cellswere injected into blood via tail vein injection at Day 4. The scaffoldswere retrieved at Day 6 and the cells in scaffolds were isolated andanalyzed in FACS (FIG. 4). In addition, the scaffolds retrieved frommice injected with fluorescent-NP-labeled leukemic cells were imagedunder fluorescent imaging instrument (Xenogel) (FIG. 5). Both resultspresented that CCL-21 released from scaffold increased the recruitmentof leukemic cells in the animal.

EXAMPLE 3 Hyperthermia-Mediated Antigen Generation

To achieve hyperthermia-mediated antigen generation from recruitedcancer cells, gold nanorods (GNRs) were incorporated in PLG macroporousscaffold (GNR-PLG scaffold) during the fabrication step (FIG. 6).GNR-PLG scaffold had 250˜440 um pores and GNRs were incorporated overthe whole scaffold, resulting in dark color in the resulting PLGscaffold. The surface of GNRs were modified with poly(ethylene glycol)(PEG) to remove the toxicity from original surfactants(cetyltrimethylammonium bromide) used in GNR-fabrication step which isknown as toxic agents to the cells.

GNRs incorporated in PLG scaffold showed intrinsic maximum absorption at˜810 nm with maximum intensity, which is desirable for in vivoirradiation due to minimum absorption by tissue and water in that rangeof wavelength (FIG. 7A). Upon irradiation with 808 nm continuous diodelaser, the temperature of GNR-PLG scaffold was increased to 40° C. (FIG.7B) from room temperature.

As described below, the temperature of GNR-PLG scaffolds were controlledin the range from room temperature up to ˜70° C. by applying differentpower of laser (FIG. 8A). By contrast, blank scaffold withoutincorporation of GNRs showed insignificant change in temperature uponirradiation with even highest power that was applied to GNR-PLGscaffold, representing the NIR-mediated hyperthermia could be inducedspecifically in GNR-incorporated scaffold. Optionally, multiple antigengeneration is used to elicit a strong immune activation. The repetitiveNIR irradiation allowed multiple hyperthermia in same GNR-PLG scaffoldwithout decrease of the targeting temperature by using same laser power(FIG. 8B).

EXAMPLE 4 GNR Absorption of NIR Light

Local hyperthermia for GNR-incorporated region of PLG scaffold waspossible due to the absorption of NIR light by GNRs in specific area ofPLG scaffold. A half GNR-PLG scaffold and a half blank PLG scaffold werephysically attached and irradiated with 808 nm laser with large beamsize to cover whole scaffold, resulting in specific heating in onlyGNR-PLG scaffold side (FIG. 9). This configuration represents a scaffoldwith different compartments, i.e., one for cancer cells and one for DCsthat can be implanted. Only the compartment for cancer cells is heated,while the one for DCs remains intact (unheated) to allow normal DCfunction.

EXAMPLE 5 A PLG Scaffold with a GNR-Incorporated Core and a Normal Shell

A single PLG scaffold composed of GNR-incorporated core part and normalshell part (FIG. 10A) was fabricated for cancer-specific heating (FIG.10B). This single PLG scaffold with different compartment andwell-interconnected pore structure (FIG. 10C) allows the efficientcancer antigen uptake by DC after hyperthermia (FIG. 10C).

EXAMPLE 6 Heat-Shocked Cancer Cells

To test if heat-shock can induce more immunogenic antigens due toadjuvant effect of HSP produced from heat-shocked cancer cells, HSPlevels of cancer cell cultures were analyzed after heat shock in 43° C.water bath (FIG. 11A). Higher HSP70, a representative HSP, were obtainedboth the cell lysate and cell culture media in heat-shocked conditioncompared with freeze-thaw method, the common lysate generating method.In vitro DC-activation by heat-shocked lysate and freeze-thaw lysateshowed that heat-shocked lysate have higher DC-activating property dueto higher HSPs (FIG. 11B).

EXAMPLE 7 NIR-Irradiation to Induce HSPs from Cancer Cells

To test if NIR-irradiation on GNR-PLG scaffold can induce HSPs fromcancer cells residing in scaffold, leukemic cells were seeded in GNR-PLGscaffold and subsequently irradiated with 808 nm laser (FIG. 12A). Thevarious temperatures by irradiation were tested for to evaluate HSP70levels, representing that irradiating to 45° C. resulted maximum HSP70level (FIG. 12B). In addition, Western blot data showed that 24 hourincubation after irradiation at 45° C. generated highest HSPs.

EXAMPLE 8 Activation of Dendritic Cells

In vitro DC, e.g., bone-marrow derived dendritic cells (BMDC),activation with cell lysates from GNR-PLG scaffold after NIR irradiationat 45° C. resulted in higher activation of DCs compared withnon-irradiated cell lysates in terms of CCR7 and CD86, therepresentative cell surface markers of activated DCs (FIG. 13),representing NIR irradiation leads to in situ generation of highlyimmunogenic cancer antigens from cancer cells residing in GNR-PLGscaffold.

EXAMPLE 9 Cancer Cell Viability After NIR Irradiation In vitro

In vitro cancer cell viability in GNR-PLG scaffold after NIR irradiationwas evaluated. Leukemic cells were seeded in GNR-PLG scaffold and NIRirradiation was applied to increase temperature to 40, 45, and 50° C.,and the viability of cells was checked with Alamar blue assay (FIG. 14).Lower cell viability resulted from higher temperature. In addition, asecond irradiation resulted in even lower viability in all conditions.These data indicate that the cancer cells were dying due to hyperthermiacaused by NIR irradiation in GNR-PLG scaffold.

EXAMPLE 10 Cancer Cell Viability After NIR Irradiation In vivo

To mimic in vivo recruitment, the GNR-PLG scaffold seeded with leukemiccells were implanted into the tissues of C57BL/6J mice subcutaneously,and the scaffolds were irradiated with NIR laser to 45 and 50° C.Similar to the in vitro experiments, the cell viability was decreased inconditions of higher temperatures (FIG. 15), indicating that theNIR-irradiation induced heat-shock to the recruited cancer cells inGNR-PLG scaffold. Recruitment of circulating cancer cells into theimplanted scaffold device, alteration or destruction of the cancer cellsby application of an external force, e.g., radiation, leads to increasedavailability of tumor antigens in the device. The increased availabilityof tumor antigens in the device for cancer leads to an increase in DCactivation and to a more effective cancer vaccine.

EXAMPLE 11 In vivo Irradiation of Implanted Device Leads to Increase inDC Activation and Number of Recruited DC's

In vivo dendritic cell recruitment and activation using laserirradiation were evaluated. 10⁶ EG7.Ova lymphoma cells were loaded intoGNR-PLG scaffolds with GM-CSF. The scaffolds were implantedsubcutaneously in C57BL/6J mouse. On day 3 post implantation thescaffold site was irradiated with 808 nm NIR laser to 45° C. for 5minutes. On day 7 post implantation, the scaffolds were retrieved,digested, and the cells were analyzed for the dendritic marker (CD11c),and the activation marker (CD86). FIGS. 16A-B show that laserirradiation significantly (n=3, p<0.05) increases the percentage ofrecruited dendritic cells (A) activates them in the scaffold (B).

EXAMPLE 12 In vivo Irradiation of Implanted Device Leads to Increase inDC Activation and Number of Activated DCs in Draining Lymph Node

In vivo dendritic cell activation in the draining lymph node using laserirradiation was evaluated. 10⁶ EG7. Ova lymphoma cells were loaded intoGNR-PLG scaffolds with GM-CSF. The scaffolds were implantedsubcutaneously in the back of a C57BL/6J mouse. On day 3 postimplantation the scaffold site was irradiated with 808 nm NIR laser to45° C. for 5 minutes. On day 7 post implantation, the draining lymphnodes (inguinal lymph nodes) were retrieved, digested, and the cellswere analyzed for the dendritic marker (CD11c), and the activationmarker (CD86). FIGS. 17A-B show that laser irradiation significantly(n=3, p<0.05) enlarges the draining lymph node (A), which is a responseafter inflammation, and increases the number of activated dendriticcells (B) in the lymph node. These data indicate that recruited DCsleave the scaffold device and migrate/relocated to draining lymph nodes(i.e., an anatomical site different from the location of the implanteddevice).

EXAMPLE 13 Irradiation as an Additional Danger Signal for Immune CellActivation

In vivo dendritic cell recruitment and activation in the full PLGvaccine using laser irradiation were evaluated. 10⁶ EG7.Ova lymphomacells were loaded into GNR-PLG scaffolds with GM-CSF and condensedCpG-ODN, which serves as the danger signal to activate DCs. Thescaffolds were implanted subcutaneously in C57BL/6J mouse. On day 3 postimplantation the scaffold site was irradiated to 45° C. for 5 minuteswith 808 nm NIR laser. On day 7 post implantation, the scaffolds wereretrieved, digested, and the cells were analyzed for the dendriticmarker (CD11c), and the activation marker (CD86). FIGS. 18A-D show thatlaser irradiation further increases (n=3, p<0.05) the percentage (A) andtotal number (B) of recruited DCs, and activates them at the scaffoldsite (C-D). This data indicate indicates that laser irradiation servesas an additional danger signal for immune cell activation.

EXAMPLE 14 Hyperthermic Treatment of Cancer Cells

In vitro BMDC activation with heat shock B16 cell lysate was evaluated.10×10⁶ B16 melanoma cells were heat shocked at 45° C. for 5 minutes inpre-warmed water bath to prepare heat-shocked cell lysate. Conventionalcell lysate was generated using 3 cycles of freeze-thaw procedure. Theprepared lysate was incubated with 10⁶ BMDCs for 18 hours. BMDCactivation markers, MHCII and CD86, were analyzed using flow cytometry.Cells were gated on CD11 c+ DCs. FIG. 19 shows that the lysate generatedfrom heat shocked cells serves as a danger signal to activate DCs,because it is capable of generating more (p<0.05) activated DCs thanconventional cell lysates.

EXAMPLE 15 Effect of Temperature on LPS Responsiveness

High temperature induces reduced responsiveness to LPS in BMDC in vitro.10⁶/well BMDCs were heat shocked at 50° C. for 5 minutes. They were thenincubated with or without LPS, an immune adjuvant capable ofupregulating immune cell activation. Activation markers, CD86 andMHC-II, were analyzed in flow cytometry. FIGS. 20 A-D show that heatshock can abrogate the BMDCs' capability to respond to LPS stimulation.Thus, an alternative scaffold structure was developed to protectrecruited DCs from irradiation.

EXAMPLE 16 Device with Core-shell Architecture

An alternative structure of GNR scaffold, a core-shell type scaffold,was engineered to avoid the direct killing of recruited BMDCs but toallow for the heat shock of recruited cancer cells. The inner corescaffold is designed to load cancer recruiting chemokine and GNR (thecolor is dark due to loaded GNR); the outer shell scaffold is loadedwith only GM-CSF to recruit DCs. Compressive testing demonstrates thatthis scaffold scheme has a lower Young's modulus than the conventionalscaffold scheme (FIGS. 21A-C). In this design, only the cancer cellsthat are recruited to the inner core scaffold are subjected to heatshock from laser irradiation and the recruited DCs in outer shellscaffold avoid heat shock.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

The invention claimed is:
 1. A biopsy-free method for producing aprocessed tumor antigen in situ comprising administering to a subjectdiagnosed with a cancer a porous 3-dimensional scaffold, said scaffoldcomprising (a) a first zone comprising a chemoattractant of cancer cellsand a cytotoxicity-inducing composition, and (b) a second zonecomprising an immune cell recruitment composition, wherein said secondzone does not comprise a cytotoxicity-inducing composition; andmaintaining said scaffold in situ for a time period sufficient toaccumulate a circulating cancer cell to yield a cancer cell-containingscaffold, and contacting said cytotoxicity-inducing composition in saidfirst zone with a cytotoxic or cytolytic element to specifically disruptsaid cancer cell, thereby producing a processed tumor antigen.
 2. Themethod of claim 1, wherein said cytotoxic element comprises applicationof external heat, ultrasound, laser radiation, or gamma radiation tosaid cell-containing scaffold.
 3. The method of claim 2, wherein saidlaser radiation comprises ultraviolet or near infrared laser radiation.4. The method of claim 1, wherein said cytotoxicity-inducing compositioncomprises a hyperthermia-inducing composition.
 5. The method of claim 4,wherein said hyperthermia-inducing composition comprises a magneticnanoparticle or a near infrared (NIR) absorbing nanoparticle.
 6. Themethod of claim 5, wherein said nanoparticle is magnetic, and whereinsaid method comprises contacting said magnetic nanoparticle with saidcytotoxic or cytolytic element to induce local hyperthermia in situ,thereby disrupting said cancer cell and producing a processed tumorantigen, wherein said cytotoxic or cytolytic element is an alternativemagnetic field.
 7. The method of claim 5, wherein said NIR nanoparticleis selected from the group consisting of a gold nanorod, gold nanoshell,gold nanocage, noble metal nanoparticle, carbon nanotube, carbonnanoparticle, and graphite nanoparticle, and wherein said methodcomprises contacting said NIR nanoparticle with said cytotoxic orcytolytic element to induce local hyperthermia in situ, therebydisrupting said cancer cell and producing a processed tumor antigen,wherein said cytotoxic or cytolytic element is NIR radiation.
 8. Themethod of claim 1, wherein said chemoattractant of cancer cellscomprises a chemokine selected from the group consisting of CCL-21,CCL-19, SDF-1, VEGF, and IL-4.
 9. The method of claim 1, wherein saidcancer is characterized by circulating tumor cells.
 10. The method ofclaim 1, wherein said subject is diagnosed with a metastatic cancercondition or a leukemia.
 11. A tumor antigen-processing devicecomprising a porous 3-dimensional scaffold, said scaffold comprising (a)a first zone comprising a chemoattractant of cancer cells and acytotoxicity-inducing composition, and (b) a second zone comprising animmune cell recruitment composition, wherein said second zone does notcomprise a cytotoxicity-inducing composition.
 12. The device of claim11, wherein said cytotoxicity-inducing composition comprises ahyperthermia-inducing particle.
 13. The device of claim 11, wherein saidcytotoxicity-inducing composition comprises a gold nanoparticle or agold nanorod.
 14. The device of claim 11, wherein said immune cellrecruitment composition comprises granulocyte macrophagecolony-stimulating factor.
 15. The device of claim 11, wherein saidfirst zone is configured as a core and said second zone is configured asa shell.
 16. The method of claim 1, further comprising maintaining saidscaffold in situ for a time period sufficient to accumulate acirculating immune cell to yield a cancer cell-and immunecell-containing scaffold prior to contacting said cytotoxicity-inducingcomposition in said first zone with a cytotoxic or cytolytic element tospecifically disrupt a cancer cell, thereby producing a processed tumorantigen.
 17. The method of claim 1, wherein said immune cell recruitmentcomposition comprises granulocyte macrophage colony-stimulating factor.18. The method of claim 17, wherein said immune cell is a dendriticcell.
 19. The method of claim 18, wherein said second zone furthercomprises a danger signal for dendritic cells.
 20. The method of claim1, wherein said first zone is configured as a core and said second zoneis configured as a shell.
 21. The method of claim 1, wherein said firstzone and said second zone are layered.
 22. The device of claim 11,wherein said first zone and said second zone are layered.
 23. The methodof claim 1, wherein said first zone comprises compartments within saidporous 3-dimensional scaffold.
 24. The device of claim 11, wherein saidfirst zone comprises compartments within said porous 3-dimensionalscaffold.